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Edson Antunes - One of the best experts on this subject based on the ideXlab platform.
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implication of rho kinase and Soluble Guanylyl Cyclase enzymes in prostate smooth muscle dysfunction in middle aged rats
Neurourology and Urodynamics, 2017Co-Authors: Fabiano B Calmasini, Fabio H Silva, Eduardo C Alexandre, Renata Lopes Rodrigues, Ana Paula L Barbosa, Danilo Lopes Ferrucci, Hernandes F Carvalho, Gabriel Forato Anhe, Andre S Pupo, Edson AntunesAbstract:Aims Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-Soluble Guanylyl Cyclase (sGC), α1- and β-adrenoceptor pathways. Methods Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [3H]-noradrenaline efflux, western blotting for α1 and β1 sGC subunits, and cyclic nucleotide levels were carried out. Results Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10−7–10−4 g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1 nM–100 μM) and α,β-methylene ATP (1–10 μM) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1–32 Hz) promoted higher [3H]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of α1 and β1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50 mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats. Conclusions Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and β-adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. © 2016 Wiley Periodicals, Inc.
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evaluation of the relaxant effect of the nitric oxide independent Soluble Guanylyl Cyclase stimulator bay 41 2272 in isolated detrusor smooth muscle
European Journal of Pharmacology, 2010Co-Authors: Fernando R Bau, Fabiola Z Monica, Fernanda B M Priviero, Lineu Baldissera, Gilberto De Nucci, Edson AntunesAbstract:Abstract The nitric oxide (NO)-independent Soluble Guanylyl Cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs–Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001–100 µM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3 ± 6.6%, 95.1 ± 9.9% and 91.7 ± 5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the Soluble Guanylyl Cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 µM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca 2+ in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca 2+ influx blockade independently of cGMP production appears to be involved.
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relaxing effects induced by the Soluble Guanylyl Cyclase stimulator bay 41 2272 in human and rabbit corpus cavernosum
European Journal of Pharmacology, 2003Co-Authors: Juliana S Baracat, Fernanda B M Priviero, Edson Antunes, Cleber E Teixeira, Cristina E Okuyama, Renato Faro, Gilberto De NucciAbstract:Abstract 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent Soluble Guanylyl Cyclase stimulator in a nitric oxide (NO)-independent manner. The relaxant effect of BAY 41-2272 was investigated in rabbit and human corpus cavernosum in vitro. BAY 41-2272 (0.01–10 μM) relaxed both rabbit (pEC50=6.82±0.06) and human (pEC50=6.12±0.10) precontracted cavernosal strips. The Guanylyl Cyclase inhibitor (ODQ, 10 μM) caused significant rightward shifts in the concentration–response curves for BAY 41-2272 in rabbit (4.7-fold) and human (2.3-fold) tissues. The NO synthesis inhibitor (N-nitro- l -arginine methyl ester ( l -NAME), 100 μM) also produced similar rightward shifts, revealing that BAY 41-2272 acts synergistically with endogenous NO to elicit its relaxant effect. The results also indicate that ODQ is selective for the NO-stimulated enzyme, since relaxations evoked by BAY 41-2272 were only partly attenuated by ODQ. The present study shows that both BAY 41-2272 and sildenafil evoke relaxations independent of inhibition of haem in Soluble guanylate Cyclase. Moreover, there is no synergistic effect of the two compounds in corpus cavernosum.
Johannespeter Stasch - One of the best experts on this subject based on the ideXlab platform.
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insights into bay 60 2770 activation and s nitrosylation dependent desensitization of Soluble Guanylyl Cyclase via crystal structures of homologous nostoc h nox domain complexes
Biochemistry, 2013Co-Authors: Vijay Kumar, Jonathan S Stamler, Faye Martin, Martina Schaefer, Johannespeter Stasch, Michael G Hahn, Focco Van Den AkkerAbstract:The Soluble Guanylyl Cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58–2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has abou...
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insights into bay 60 2770 activation and s nitrosylation dependent desensitization of Soluble Guanylyl Cyclase via crystal structures of homologous nostoc h nox domain complexes
Biochemistry, 2013Co-Authors: Vijay Kumar, Jonathan S Stamler, Faye Martin, Martina Schaefer, Johannespeter Stasch, Michael Hahn, Focco Van Den AkkerAbstract:The Soluble Guanylyl Cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.
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structure of cinaciguat bay 58 2667 bound to nostoc h nox domain reveals insights into heme mimetic activation of the Soluble Guanylyl Cyclase
Journal of Biological Chemistry, 2010Co-Authors: Annie Beuve, Padmamalini Baskaran, Faye Martin, Pete W Dunten, Martina Schaefer, Johannespeter Stasch, Focco Van Den AkkerAbstract:Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of Soluble Guanylyl Cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58–2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58–2667. The 2.3-Å resolution structure of BAY 58–2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58–2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58–2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58–2667 binding causes a rotation of the αF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His105–heme covalent bond. The structure provides insights into how BAY 58–2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.
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distinct molecular requirements for activation or stabilization of Soluble Guanylyl Cyclase upon haem oxidation induced degradation
BMC Pharmacology, 2009Co-Authors: Harald H H W Schmidt, Johannespeter Stasch, Linda S Hoffmann, Peter M Schmidt, Yvonne Keim, Stefan SchaeferAbstract:In endothelial dysfunction, signalling by nitric oxide(NO) is impaired because of the oxidation and subse-quent loss of the Soluble Guanylyl Cyclase (sGC) haem [1].The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethyl-benzyl)oxy]phenethyl}amino)methyl [benzoic]acid(BAY 58-2667) is a haem-mimetic able to bind with highaffinity to GC when the native haem (the NO bindingsite) is removed and it also protects sGC from ubiquitin-triggered degradation [2-4]. Here we investigate whetherthis protection is a unique feature of BAY 58-2667 or ageneral characteristic of haem-site ligands such as thehaem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-
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distinct molecular requirements for activation or stabilization of Soluble Guanylyl Cyclase upon haem oxidation induced degradation
British Journal of Pharmacology, 2009Co-Authors: Harald H H W Schmidt, Johannespeter Stasch, Linda S Hoffmann, Peter M Schmidt, Yvonne Keim, Stefan SchaeferAbstract:Background and purpose: In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the Soluble Guanylyl Cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272). Experimental approach: The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed. Key results: Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant β1H105F was stabilized by BAY 58-2667 and Zn-PPIX. Conclusions: The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.
Gilberto De Nucci - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the relaxant effect of the nitric oxide independent Soluble Guanylyl Cyclase stimulator bay 41 2272 in isolated detrusor smooth muscle
European Journal of Pharmacology, 2010Co-Authors: Fernando R Bau, Fabiola Z Monica, Fernanda B M Priviero, Lineu Baldissera, Gilberto De Nucci, Edson AntunesAbstract:Abstract The nitric oxide (NO)-independent Soluble Guanylyl Cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs–Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001–100 µM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3 ± 6.6%, 95.1 ± 9.9% and 91.7 ± 5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the Soluble Guanylyl Cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 µM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca 2+ in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca 2+ influx blockade independently of cGMP production appears to be involved.
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relaxing effects induced by the Soluble Guanylyl Cyclase stimulator bay 41 2272 in human and rabbit corpus cavernosum
European Journal of Pharmacology, 2003Co-Authors: Juliana S Baracat, Fernanda B M Priviero, Edson Antunes, Cleber E Teixeira, Cristina E Okuyama, Renato Faro, Gilberto De NucciAbstract:Abstract 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent Soluble Guanylyl Cyclase stimulator in a nitric oxide (NO)-independent manner. The relaxant effect of BAY 41-2272 was investigated in rabbit and human corpus cavernosum in vitro. BAY 41-2272 (0.01–10 μM) relaxed both rabbit (pEC50=6.82±0.06) and human (pEC50=6.12±0.10) precontracted cavernosal strips. The Guanylyl Cyclase inhibitor (ODQ, 10 μM) caused significant rightward shifts in the concentration–response curves for BAY 41-2272 in rabbit (4.7-fold) and human (2.3-fold) tissues. The NO synthesis inhibitor (N-nitro- l -arginine methyl ester ( l -NAME), 100 μM) also produced similar rightward shifts, revealing that BAY 41-2272 acts synergistically with endogenous NO to elicit its relaxant effect. The results also indicate that ODQ is selective for the NO-stimulated enzyme, since relaxations evoked by BAY 41-2272 were only partly attenuated by ODQ. The present study shows that both BAY 41-2272 and sildenafil evoke relaxations independent of inhibition of haem in Soluble guanylate Cyclase. Moreover, there is no synergistic effect of the two compounds in corpus cavernosum.
Harald H H W Schmidt - One of the best experts on this subject based on the ideXlab platform.
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distinct molecular requirements for activation or stabilization of Soluble Guanylyl Cyclase upon haem oxidation induced degradation
BMC Pharmacology, 2009Co-Authors: Harald H H W Schmidt, Johannespeter Stasch, Linda S Hoffmann, Peter M Schmidt, Yvonne Keim, Stefan SchaeferAbstract:In endothelial dysfunction, signalling by nitric oxide(NO) is impaired because of the oxidation and subse-quent loss of the Soluble Guanylyl Cyclase (sGC) haem [1].The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethyl-benzyl)oxy]phenethyl}amino)methyl [benzoic]acid(BAY 58-2667) is a haem-mimetic able to bind with highaffinity to GC when the native haem (the NO bindingsite) is removed and it also protects sGC from ubiquitin-triggered degradation [2-4]. Here we investigate whetherthis protection is a unique feature of BAY 58-2667 or ageneral characteristic of haem-site ligands such as thehaem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-
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distinct molecular requirements for activation or stabilization of Soluble Guanylyl Cyclase upon haem oxidation induced degradation
British Journal of Pharmacology, 2009Co-Authors: Harald H H W Schmidt, Johannespeter Stasch, Linda S Hoffmann, Peter M Schmidt, Yvonne Keim, Stefan SchaeferAbstract:Background and purpose: In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the Soluble Guanylyl Cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272). Experimental approach: The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed. Key results: Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant β1H105F was stabilized by BAY 58-2667 and Zn-PPIX. Conclusions: The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.
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immunohistochemical localization of nitric oxide synthase and Soluble Guanylyl Cyclase in the ventral cochlear nucleus of the rat
The Journal of Comparative Neurology, 2001Co-Authors: Alain C Burette, Harald H H W Schmidt, Peter Petrusz, Richard J WeinbergAbstract:The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of Soluble Guanylyl Cyclase (sGC), a heterodimer comprised of alpha and beta subunits. The authors used immunohistochemistry to study the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague-Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS-I) but not for sGCbeta. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS-I and sGCbeta; these cells could be seen at times receiving contacts from NOS-I-positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS-I was concentrated mainly in somata. Double-labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS-I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS-I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L-citrulline (a by-product of the formation of NO from L-arginine) and cGMP, respectively. L-citrulline was restricted to NOS-I-positive elements, and the large majority of NOS-expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC-positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner.
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the Soluble Guanylyl Cyclase inhibitor 1h 1 2 4 oxadiazolo 4 3 a quinoxalin 1 one is a nonselective heme protein inhibitor of nitric oxide synthase and other cytochrome p 450 enzymes involved in nitric oxide donor bioactivation
Molecular Pharmacology, 1999Co-Authors: Martin Feelisch, Peter Kotsonis, Jan Siebe, Bernd Clement, Harald H H W SchmidtAbstract:Soluble Guanylyl Cyclase (sGC) is an important effector for nitric oxide (NO). It acts by increasing intracellular cyclic GMP (cGMP) levels to mediate numerous biological functions. Recently, 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) was identified as a novel and selective inhibitor of this enzyme. Therefore, ODQ may represent an important pharmacological tool for differentiating cGMP-mediated from cGMP-independent effects of NO. In the present study, we examined the inhibitory action of ODQ both functionally and biochemically. In phenylephrine-preconstricted, endothelium-intact, isolated aortic rings from the rat, ODQ, in a concentration-dependent manner, increased contractile tone and inhibited relaxations to authentic NO with maximal effects at 3 microM. Pretreatment of vascular rings with ODQ induced a parallel, 2-log-order shift to the right of the concentration-response curves (CRCs) to histamine, ATP, NO, the NO-donors S-nitrosoglutathione, S-nitroso-N-acetyl-D,L-penicillamine, and spermine NONOate [N-[4-[1-(3-amino propyl)-2-hydroxy-2-nitroso hydrazino]butyl]-1, 3-propane diamine], and the direct sGC-stimulant [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] YC-1 but did not affect relaxations induced by papaverine and atriopeptin II. Moreover, the rightward shift of the CRCs to Angeli's salt, peroxynitrite, and linsidomine was similar to that of NO. These results suggested that ODQ is specific for sGC. Furthermore, they indicate that NO can cause vasorelaxation independent of cGMP. Three interesting exceptions were observed to the otherwise rather uniform inhibitory effect of ODQ: the responses to acetylcholine, glycerol trinitrate, and sodium nitroprusside. The latter two agents are known to require metabolic activation, possibly by cytochrome P-450-type proteins. The 3- to 5-log-order rightward shift of their CRCs suggests that, in addition to sGC, ODQ may interfere with heme proteins involved in the bioactivation of these NO donors and the mechanism of vasorelaxation mediated by acetylcholine. In support of this notion, ODQ inhibited hepatic microsomal NO production from both glycerol trinitrate and sodium nitroprusside as well as NO synthase activity in aortic homogenates. The latter effect seemed to require biotransformation of ODQ. Collectively, these data reveal that ODQ interferes with various heme protein-dependent processes in vascular and hepatic tissue and lacks specificity for sGC.
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mapping of neural nitric oxide synthase in the rat suggests frequent co localization with nadph diaphorase but not with Soluble Guanylyl Cyclase and novel paraneural functions for nitrinergic signal transduction
Journal of Histochemistry and Cytochemistry, 1992Co-Authors: Harald H H W Schmidt, Gerard D Gagne, Masaki Nakane, Jennifer S Pollock, Mahlon F Miller, F MuradAbstract:Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates Soluble Guanylyl Cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most wide...
Focco Van Den Akker - One of the best experts on this subject based on the ideXlab platform.
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insights into bay 60 2770 activation and s nitrosylation dependent desensitization of Soluble Guanylyl Cyclase via crystal structures of homologous nostoc h nox domain complexes
Biochemistry, 2013Co-Authors: Vijay Kumar, Jonathan S Stamler, Faye Martin, Martina Schaefer, Johannespeter Stasch, Michael G Hahn, Focco Van Den AkkerAbstract:The Soluble Guanylyl Cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58–2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has abou...
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structure of cinaciguat bay 58 2667 bound to nostoc h nox domain reveals insights into heme mimetic activation of the Soluble Guanylyl Cyclase
Journal of Biological Chemistry, 2010Co-Authors: Annie Beuve, Padmamalini Baskaran, Faye Martin, Pete W Dunten, Martina Schaefer, Johannespeter Stasch, Focco Van Den AkkerAbstract:Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of Soluble Guanylyl Cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58–2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58–2667. The 2.3-Å resolution structure of BAY 58–2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58–2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58–2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58–2667 binding causes a rotation of the αF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His105–heme covalent bond. The structure provides insights into how BAY 58–2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.